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[Xia Li](https://orcid.org/0000-0002-3246-4462), [Shinya Hattori](https://orcid.org/0000-0002-2635-2464), [Mitsuhiro Ebara](https://orcid.org/0000-0002-7906-0350), [Naoto Shirahata](https://orcid.org/0000-0002-1217-7589), [Nobutaka Hanagata](https://orcid.org/0000-0001-6079-3426)

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[A facile approach to preparing personalized cancer vaccines using iron-based metal organic framework](https://mdr.nims.go.jp/datasets/0ee96c6f-6c1c-4f8b-8477-1f7af87fd589)

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A facile approach to preparing personalized cancer vaccines using iron-based metal organic frameworkFrontiers in ImmunologyOPEN ACCESSEDITED BYFernando Torres Andón,Institute of Biomedical Research of A Coruña(INIBIC), SpainREVIEWED BYMing Yi,Zhejiang University, ChinaYao-Xin Lin,Chinese Academy of Sciences (CAS), China*CORRESPONDENCEXia LiLI.Xia@nims.go.jpRECEIVED 29 October 2023ACCEPTED 14 December 2023PUBLISHED 08 January 2024CITATIONLi X, Hattori S, Ebara M, Shirahata N andHanagata N (2024) A facile approach topreparing personalized cancer vaccines usingiron-based metal organic framework.Front. Immunol. 14:1328379.doi: 10.3389/fimmu.2023.1328379COPYRIGHT© 2024 Li, Hattori, Ebara, Shirahata andHanagata. This is an open-access articledistributed under the terms of the CreativeCommons Attribution License (CC BY). 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No use,distribution or reproduction is permittedwhich does not comply with these terms.TYPE Original ResearchPUBLISHED 08 January 2024DOI 10.3389/fimmu.2023.1328379A facile approach to preparingpersonalized cancer vaccinesusing iron-based metalorganic frameworkXia Li1*, Shinya Hattori2, Mitsuhiro Ebara1, Naoto Shirahata3,4and Nobutaka Hanagata11Research Center for Macromolecules and Biomaterials, National Institute for Materials Science(NIMS), Tsukuba, Ibaraki, Japan, 2Bioanalysis Unit, Research Network and Facility Services Division,National Institute for Materials Science (NIMS), Tsukuba, Ibaraki, Japan, 3Research Center for MaterialsNanoarchitectonics (MANA), National Institute for Materials Science (NIMS), Tsukuba, Ibaraki, Japan,4Graduate School of Chemical Sciences and Engineering, Hokkaido University, Sapporo, JapanBackground: Considering the diversity of tumors, it is of great significance todevelop a simple, effective, and low-cost method to prepare personalizedcancer vaccines.Methods: In this study, a facile one-pot synthetic route was developed to preparecancer vaccines using model antigen or autologous tumor antigens based on thecoordination interaction between Fe3+ ions and endogenous fumarate ligands.Results: Herein, Fe-based metal organic framework can effectively encapsulatetumor antigens with high loading efficiency more than 80%, and act as bothdelivery system and adjuvants for tumor antigens. By adjusting the synthesisparameters, the obtained cancer vaccines are easily tailored from microscalerod-like morphology with lengths of about 0.8 mm (OVA-ML) to nanoscalemorphology with sizes of about 50~80 nm (OVA-MS). When cocultured withantigen-presenting cells, nanoscale cancer vaccines more effectively enhanceantigen uptake and Th1 cytokine secretion than microscale ones. Nanoscalecancer vaccines (OVA-MS, dLLC-MS) more effectively enhance lymph nodetargeting and cross-presentation of tumor antigens, mount antitumorimmunity, and inhibit the growth of established tumor in tumor-bearing mice,compared with microscale cancer vaccines (OVA-ML, dLLC-ML) and freetumor antigens.Conclusions: Our work paves the ways for a facile, rapid, and low-costpreparation approach for personalized cancer vaccines.KEYWORDScancer immunotherapy, personalized cancer vaccines, metal organic framework, ferricions, endogenous fumarate ligands, nanotechnology, adjuvantsfrontiersin.org01https://www.frontiersin.org/articles/10.3389/fimmu.2023.1328379/fullhttps://www.frontiersin.org/articles/10.3389/fimmu.2023.1328379/fullhttps://www.frontiersin.org/articles/10.3389/fimmu.2023.1328379/fullhttps://www.frontiersin.org/articles/10.3389/fimmu.2023.1328379/fullhttps://www.frontiersin.org/journals/immunologyhttps://www.frontiersin.orghttp://crossmark.crossref.org/dialog/?doi=10.3389/fimmu.2023.1328379&domain=pdf&date_stamp=2024-01-08mailto:LI.Xia@nims.go.jphttps://doi.org/10.3389/fimmu.2023.1328379http://creativecommons.org/licenses/by/4.0/http://creativecommons.org/licenses/by/4.0/https://www.frontiersin.org/journals/immunology#editorial-boardhttps://www.frontiersin.org/journals/immunology#editorial-boardhttps://doi.org/10.3389/fimmu.2023.1328379https://www.frontiersin.org/journals/immunologyLi et al. 10.3389/fimmu.2023.13283791 IntroductionIn the past decade, cancer immunotherapy has been in thespotlight as an innovative technology that alters the direction ofcancer treatment (1–5). Among them, immune checkpointinhibitors have been approved as first-line drugs in the treatmentof diverse cancers (4). However, the response rate for immunecheckpoint inhibitor monotherapy remains at 10-40%, because theprerequisite for its effectiveness is the preexistence of large amountsof tumor antigen-specific T cells (4, 6). Therefore, therapeuticcancer vaccines that induce tumor antigen-specific T-cell immuneresponse are expected to be the next breakthrough in cancerimmunotherapy (1–3, 7).Therapeutic cancer vaccines aim to train the patients’ ownimmune system to recognize and eradicate cancer cells in the body(1–3). Tumor antigens are antigenic substances produced by tumorcells, which are tumor markers to identify and recognize tumorcells. According to the source of tumor antigens, cancer vaccinescan be divided into two categories: shared tumor antigensvaccines and personalized tumor antigens vaccines (8–10). Cancervaccines made from shared antigens (e.g., free peptides) had beenthe mainstream of clinical research since the 1990s, but their clinicalresponse rates were low, due to tumor heterogeneity, insufficientimmunogenicity, susceptibility to tumor antigen loss, and the lackof effective adjuvants (8, 11). Compared with shared tumor antigensvaccines, personalized tumor antigen vaccines generally exhibitedmuch higher response rates (8–10). Personalized tumor antigensvaccines are further classified into predefined personalized antigensvaccines and unidentified personalized antigens vaccines (1–3).Predefined personalized antigens vaccines (e.g., neoantigenvaccines) are associated with extremely high cost, extremely time-consuming preparation process and immune escape ofheterogeneous tumors (1–3). While, unidentified personalizedantigens vaccines derived from lysed tumor cells may greatlylower the cost of personalized cancer vaccines, and are expectedto be less susceptible to tumor antigen loss because they carry largenumbers of diverse tumor antigens (8–10).On the other hand, adjuvants play an important role inenhancing the immunogenicity of tumor antigens and thetherapeutic efficacy of cancer vaccines (12–19). Especially,codelivery of antigens and adjuvants in cancer vaccines areexpected to trigger a robust antitumor immunity and develophighly efficient cancer vaccines (12, 14, 20, 21). Despitetremendous efforts in the past decades, it remains difficult toprepare personalized cancer vaccines using a facile approach withuniversal adjuvants and high loading efficiency.Fumaric acid is an endogenous molecule in the human body,since it is an intermediate in the citric acid cycle, which cells use togenerate energy from food in the form of adenosine triphosphate(ATP), and is also a product of the urea cycle (22). Fumarate iswidely used in food additives, such as acid regulator, andpharmaceuticals. For example, ferrous fumarate is clinically usedto treat iron deficiency anemia (23). Dimethyl fumarate is a drugclinically used to treat the autoimmune diseases psoriasis andmultiple sclerosis (24). Tenofovir disoproxil fumarate is anantiviral drug approved by the United States Food and DrugFrontiers in Immunology 02Administration (FDA) for the treatment of chronic hepatitis Bvirus infection (HBV) and human immunodeficiency virusinfection (HIV) (25). Recently, dimethyl fumarate is reported tobe highly cytotoxic in cancer cells with KRAS mutation, one of themost common molecular alterations in adult carcinomas (26). In2023, fumarate is reported to induce the release of mitochondrialDNA into the cytosol, stimulate interferon production, and driveinnate immunity (27), which may be associated with immuneinfiltration in cold tumors. On the other hand, iron elements helpstrengthen the immune system (28, 29).In this study, a facile, rapid, and low-cost one-pot route wasdeveloped to prepare personalized cancer vaccines by embeddingmodel antigen or autologous tumor antigens within Fe-based metalorganic framework through the coordination interaction betweenFe3+ ions and endogenous fumarate ligands. Herein, Fe-based metalorganic framework can effectively encapsulate tumor antigens withhigh loading efficiency >80%, and act as both delivery system andadjuvants for tumor antigens. By adjusting the synthesisparameters, the morphology of the obtained cancer vaccines istailored from microscale to nanoscale. Personalized cancer vaccineseffectively enhance antigen uptake and Th1 cytokine secretion,strengthen lymph node targeting and cross-presentation of tumorantigens, mount antitumor immunity, and inhibit the growth ofestablished tumor in tumor-bearing mice.2 Materials and methods2.1 MaterialsIron (III) chloride hexahydrate (FeCl3·6H2O), fumaric acid(C4H4O4) and sodium hydroxide (NaOH) were purchased fromFujifilm Wako, Japan. Ovalbumin (OVA) was purchased fromSigma-Aldrich.2.2 Synthesis of metal organic frameworkand cancer vaccineIn a typical synthesis, metal organic framework was prepared bymixing fumaric acid (100 mM, 300 mL), NaOH solution (1 M, 30mL), FeCl3·6H2O solution (100 mM, 300~600 mL), and water tomake the total volume 3 mL with sonication for 30 min, 2 hours, or4 hours in ice. The resulting products were centrifuged, washed withultrapure water, dispersed in water for later use or freeze-dried.Lewis lung carcinoma cells (LLC, Bioresource Research Center,Japan) at 6.7×106 cells/mL were repeatedly frozen and thawed 4times in minus 30 degrees refrigerator and ice to prepare tumor celllysate, and then centrifuged at 1500 rpm for 3 min to obtain thesupernatant, which was named autologous tumor antigen dLLC.In a typical synthesis of cancer vaccines, model antigen OVA(50 mg/mL, 12 uL) or autologous tumor antigen dLLC (36 uL) wasencapsulated into metal organic framework by mixing fumaric acid(100 mM, 300 mL), NaOH solution (1 M, 30 mL), FeCl3·6H2Osolution (100 mM, 300~600 mL), and water to make the totalvolume 3 mL with sonication for 30 min or 2 hours in ice.frontiersin.orghttps://doi.org/10.3389/fimmu.2023.1328379https://www.frontiersin.org/journals/immunologyhttps://www.frontiersin.orgLi et al. 10.3389/fimmu.2023.13283792.3 Quantitative approach of biomoleculesloading amounts and samples massThe concentrations of model antigen OVA or LLC tumor celllysate in solutions before and after loading are analyzed using aMicro BCA protein assay kit (Thermo Scientific Inc.). Theencapsulation efficiencies of proteins are calculated by thefollowing formula, respectively: Proteins encapsulation efficiency= (Initial concentration - Final concentration after encapsulation)/Initial concentration×100%. The mass of metal organic frameworkor cancer vaccines is calculated by measuring weight of tubes beforeand after synthesis reaction.2.4 Physicochemical characterizationMorphological observation was carried out using field emissionhigh resolution scanning electron microscope (FE-SEM, HitachiSU8000, Japan) after being coated with platinum or carbon. Theanalysis of phases was conducted by a powder X-ray diffractometerwith CuKa X-ray (RINT-Ultima III, Rigaku, Japan). The sampleswere analyzed using Fourier transform infrared spectroscopy(IRTracer-100, Shimadzu, Japan). The zeta potentials weredetermined using a zeta potential analyzer (ELSZ-1000Z, OtsukaElectronics, Japan). The hydrodynamic diameters were measuredusing a dynamic light scattering spectrophotometer (DLS-8000HAL, Otsuka Electronics, Japan).2.5 Cellular uptake of OVA antigen andDCs activation in vitroBone marrow derived DCs were harvested as follows (7). Firstly,bone marrow cells were collected from femurs and tibias of mice(C57BL/6J, CLEA Inc.). After red blood cell lysis and depletion of I-A/I-E-, CD4- and CD8-expressing cells, the residual cells werecultured in RPMI 1640 medium supplemented with 10% fetalbovine serum (FBS) and 20 ng/mL granulocyte macrophagecolony-stimulating factor (GM-CSF). The nonadherent andloosely adherent cells were collected as bone marrow derived DCson day 9-10.DCs were seeded at a density of 5×104 cell/cm2 in a glass bottomdish for several hours. Then, cancer vaccines synthesized usingfluorescein conjugated- ovalbumin (F-OVA, Life technologies) wereadded into the above DCs medium at a final concentration of 30 mg/mL for particle and 5 mg/mL for F-OVA. F-OVA in free format withan equivalent dose was used as control. After overnight culture, cellswere stained with lysosome marker (LysoTracker Red) and nuclearstaining dye (Hoechst), added with ProLong Live Antifade Reagentto prevent the loss of fluorescent signal due to photobleaching, andobserved using a confocal laser scanning microscope (CLSM, LeicaTCS SP5).To further analyze their activation, DCs were seeded onto a flat-bottom 96-well cell culture plate at 2×105 cells/well and thenexposed to cancer vaccines suspensions with a particleconcentration of 30 mg/mL and an OVA concentration of 5 mg/Frontiers in Immunology 03mL, respectively. OVA in free format was used as control. One daylater, the supernatant was collected to quantify the cytokinesconcentration using enzyme-linked immunosorbent assay kit(ELISA, BD Biosciences).2.6 Lymph node targeting and antigencross-presentation in vivoFemale C57BL/6J mice (CLEA Inc.) were immunized bysubcutaneously injecting Fe- based cancer vaccines into the leftflank (Fe-based metal organic framework particles, 600 mg/mouse;F-OVA, 100 mg/mouse). An equivalent dose of F-OVA in freeformat was used as control. Immunized mice were euthanized oneday later, and nearby draining lymph nodes were harvested. Toanalyze the lymph node targeting, the obtained lymph nodes werefreshly frozen in Tissue-Tek O.C.T. compound to prepare thecryostat sections. Then, the sections were mounted usingSlowFade™ Diamond mountant with DAPI and observed usingLeica CLSM. To carry out the analysis about antigen cross-presentation, the obtained lymph nodes were ground through a70 mm cell strainer to obtain single cell suspension. The obtainedcells were blocked with purified anti-CD16/CD32 antibody, andthen stained with anti-CD11c-APC and anti-H-2Kb- SIINFEKL-PE(BioLegend) antibodies. Flow cytometry was performed using aSpectral Cell Analyzer (SP6800, Sony). FlowJo software was used forthe analysis of flow cytometry data.2.7 Anti-tumor experiments using E.G7-OVA lymphoma in vivoTwenty female C57BL/6J mice (5~6 weeks old, CLEA Inc.) wererandomly divided into four groups. First, E.G7-OVA lymphomacells (American Type Culture Collection, ATCC; 1.2×105 cells/mouse) were subcutaneously inoculated into the left flanks ofmice on day 0. On days 4, 7, and 10 post tumor inoculation, thefollowing substances in 100mL saline were subcutaneously injectedinto the right flanks of mice according to the divided groups:1) saline; 2) OVA (100 mg/mouse OVA in free format); 3) OVA-ML (Large-size metal organic framework encapsulated with OVA:100 mg/mouse OVA and 600 mg/mouse particles); 4) OVA-MS(Small-size metal organic framework encapsulated with OVA:100 mg/mouse OVA and 600 mg/mouse particles). Tumor size wasmeasured by a caliper and tumor volume was calculated accordingto the formula: 1/2 × length × width2.At the endpoint, spleen was harvested and triturated through a70 mm cell strainer to obtain single cell suspension. The cells werestained with anti-CD4-FITC (Biolegend), anti-CD8a-APC/Cyanine7 (Biolegend) and T-Select H-2Kb OVA Tetramer-SIINFEKL-APC (MBL) antibodies after the Fc block usingpurified anti-CD16/CD32 antibody. Flow cytometry wasperformed using a Spectral Cell Analyzer (SP6800, Sony) anddata analysis was carried out with FlowJo software. In addition,the spleen was digested with a tissue protein extraction reagent(Thermo Scientific Inc.) at the same ratio of the tissue weight tofrontiersin.orghttps://doi.org/10.3389/fimmu.2023.1328379https://www.frontiersin.org/journals/immunologyhttps://www.frontiersin.orgLi et al. 10.3389/fimmu.2023.1328379extraction reagent, and cytokines contents were determined byELISA kits (BD Biosciences).2.8 Anti-tumor experiments using Lewislung carcinoma in vivoFemale C57BL/6J mice (5~6 weeks old, CLEA Inc.) weresubcutaneously inoculated with Lewis lung carcinoma cells (8×104cells/mouse) into their left flanks on day 0. On days 4, 7, and 10 posttumor inoculation, the following substances in 100mL saline weresubcutaneously injected into the right flanks of mice according tothe divided groups: 1) saline; 2) dLLC (6 mL autologous tumorantigens/mouse in free format); 3) dLLC-ML (Large-size metalorganic framework encapsulated with autologous tumor antigens:6 mL/mouse autologous tumor antigens and 600 mg/mouseparticles); 4) dLLC-MS (Small-size metal organic frameworkencapsulated with autologous tumor antigens: 6 mL/mouseautologous tumor antigens and 600 mg/mouse particles). Tumorsize was measured by a caliper and tumor volume was calculatedaccording to the formula: 1/2 × length × width2.At the endpoint, spleen was harvested and triturated through a70 mm cell strainer to obtain single cell suspension. The cells werestained with anti-CD3-APC, anti-CD4-PE/Cyanine7, anti-CD8a-APC/Cyanine7, anti-CD44-FITC and anti-CD62L-PE antibodies(Biolegend) after the Fc block using purified anti-CD16/CD32antibody. Flow cytometry was performed using a Spectral CellAnalyzer (SP6800, Sony) and data analysis was carried out withFlowJo software.2.9 Statistical analysisAll data are presented as the mean ± standard deviation (SD).Data were analyzed using one-way analysis of variance (ANOVA)followed by Tukey’s multiple comparisons post hoc test. A p valueless than or equal to 0.05 is considered statistically significant.2.10 Ethical issueAll the animal experiments included in this study have beenapproved by the Animal Ethics Committee of National Institute forMaterials Science (NIMS), Japan. All the animal experimentalprocedures and animal care were performed in accordance withthe guidelines of the Animal Ethics Committee of NIMS, Japan.3 Results3.1 One-pot synthesis of FeMOF-basedcancer vaccinesFe-based metal organic framework (FeMOF) was synthesizedby mixing fumaric acid, NaOH solution, FeCl3·6H2O solution, andFrontiers in Immunology 04water with sonication in ice. To prepare personalized cancervaccines, OVA model antigens or autologous LLC lysates weresupplemented and encapsulated into FeMOF during the synthesisprocess. Energy dispersive X-ray (EDX) mapping analysis of cancervaccines suggest that S elements -containing OVA model antigensare homogeneously embedded in the formed FeMOFparticles (Figure 1A).The obtained cancer vaccines ranged from microscale tonanoscale by adjusting the synthesis parameter, such asconcentration of raw materials (Figure 1B). FeMOF-based cancervaccines with OVA model antigens, synthesized in ice with the finalratios of FeCl3·6H2O and fumaric acid at 1:1 for 30 min and 2 hours,exhibit nanoparticle-like morphology with sizes of about 50~80 nmand rod-like morphology with lengths of about 0.8 mm, respectively,which are named OVA-MS and OVA-ML, respectively. When thereaction time is further extended to 4 hours, FeMOF-based cancervaccines show the same rod-like morphology of about 0.8 mm asthat of 2 hours. When the final ratios of FeCl3·6H2O and fumaricacid were changed from 1:1 to 2:1, FeMOF-based cancer vaccinessynthesized in ice for 4 hours exhibit rod-like morphology withlengths of about 1.5 mm.Fourier transform infrared spectroscopy (FTIR) confirms theformation of Fe-fumarate coordination compounds and theencapsulation of OVA model antigens within them (Figure 1C).Fumaric acid shows strong C=O stretching band near 1655 cm-1,which is attributed to its carboxylic acid group. FeMOF sampleswithout the presence of antigens, including MS and ML, exhibit theabsorption bands near 1585 cm-1 and 1382 cm-1, which areattributed to asymmetric and symmetric stretching modes in thecarboxyl group of metal fumarates (Figure 1C, Left). FeMOF-basedcancer vaccines with OVA model antigens, including OVA-MS andOVA-ML, show similar absorption bands near 1585 cm-1 and 1382cm-1 with FeMOF, suggesting the formation of metal fumarates(Figure 1C, Right). In addition, the shoulder absorption bands near1635 cm-1 in OVA-MS and OVA-ML suggest the encapsulation ofOVA model antigens in FeMOF-based cancer vaccines, since OVAmodel antigens show strong C=O stretching bands near 1635 cm-1(Figure 1C, Right).Wide-angle X-ray powder diffraction (XRD) patterns of FeMOFand FeMOF-based cancer vaccines with different sizes indicate theformation of Fe-based metal organic framework MIL-88A(Figure 1D). The zeta potentials of FeMOF-based cancer vaccinesOVA-MS and OVA-ML are centered at 21 and 27 mV, respectively(Figure 1E). Dynamic light scattering (DLS) measurements suggestthat the hydrodynamic sizes of OVA-MS and OVA-ML areapproximately 300 nm and 3 mm, respectively (Figure 1F), whichis larger than the particle size directly observed by SEM due to thepartial aggregation of particles in the solution without dispersant.The concentrations of model antigen OVA or LLC tumor celllysate in solutions before and after loading into FeMOFwere analyzedusing a Micro BCA protein assay kit. The encapsulation efficiencies ofOVA in FeMOF-based cancer vaccines OVA-MS and OVA-ML areabout 90% and 91%, respectively. While the encapsulation efficienciesof autologous LLC lysates in FeMOF-based cancer vaccines dLLC-MSand dLLC -ML are about 80% and 82%, respectively.frontiersin.orghttps://doi.org/10.3389/fimmu.2023.1328379https://www.frontiersin.org/journals/immunologyhttps://www.frontiersin.orgLi et al. 10.3389/fimmu.2023.13283793.2 Size-dependent antigen uptake by DCsand their activation in vitroDCs are the most professional antigen-presenting cells touptake tumors antigens and trigger an adaptive immuneresponse. In this study, primary bone marrow derived DCs frommice were cocultured with as-prepared cancer vaccines overnight totest the cellular uptake of antigens and antigen-presenting cellsactivation. Herein, fluorescein conjugated - ovalbumin (F-OVA)with green fluorescence was used to prepare cancer vaccines withvisualized antigens. F-OVA solutions in free format was used ascontrol group. Small-size cancer vaccines (OVA-MS) present muchhigher green fluorescence intensity than large-size cancer vaccines(OVA-ML) and free F-OVA control group, as shown in CLSMFrontiers in Immunology 05images (Figure 2A). The images suggest that F-OVA moleculesembedded in small-size particles can be more effectively captured byDCs than those embedded in large-size particles and those infree format.Further, when DCs were cocultured with cancer vaccinesembedded with OVA model tumor antigens for one day, theiractivation was assessed using ELISA assay (Figure 2B). DCscocultured with FeMOF-based cancer vaccines show significantlyhigher cytokines secretion, such as interleukin (IL) -1b and tumornecrosis factor (TNF) -a, compared with free OVA and mediumgroups. DCs cocultured with small-size cancer vaccines moreefficiently promote the cytokines secretion, including IL-12 andinterferon (IFN) -g, compared with large-size cancer vaccines, freeOVA, and medium groups.BC DE FAFIGURE 1Physicochemical characterization of FeMOF and FeMOF-based cancer vaccines with tailored size. (A) EDX mapping analysis of FeMOF-based cancervaccine (scale bar 1mm). Uniform distribution of Fe, O and S elements evidenced that model antigen OVA was homogeneously embedded in the FeMOFpartilces. (B) FeMOF-based cancer vaccines with OVA model antigens synthesized in the ratio of Fe3+ and fumaric acid at 1:1 for 30 min (OVA-MS, Left 1,scale bar 500nm), 2 hours (OVA-ML, Left 2, scale bar 1mm), and 4 hours (Left 3, scale bar 1mm). FeMOF-based cancer vaccines with OVA model antigenssynthesized in the ratio of Fe3+ and fumaric acid at 2:1 for 4 hours (Left 4, scale bar 1mm). (C) FTIR spectra of fumaric acid and FeMOF with different sizes(MS, ML) (Left). FTIR spectra of OVA model antigens and FeMOF -based cancer vaccines with OVA model antigens (OVA-MS, OVA-ML) (Right). (D) XRDpatterns of FeMOF and FeMOF-based cancer vaccines. (E) Zeta potentials of FeMOF -based cancer vaccines. (F) Particle size distribution of samplesFeMOF -based cancer vaccines.frontiersin.orghttps://doi.org/10.3389/fimmu.2023.1328379https://www.frontiersin.org/journals/immunologyhttps://www.frontiersin.orgLi et al. 10.3389/fimmu.2023.13283793.3 FeMOF-based cancer vaccines in smallsize significantly strengthen lymph nodetargeting and cross-presentation of tumorantigens in vivoTo test the lymph node targeting abilities of tumor antigens indifferent vaccine formulations in vivo, the obtained vaccinessynthesized using fluorescein conjugated- OVA model antigenswith green fluorescence were subcutaneously injected into C57BL/6J mice, and the draining lymph nodes were collected 16 hours later.Then the cryosections of the draining lymph nodes were preparedand observed by CLSM. As shown in Figure 3A, small-size cancervaccines groups (OVA-MS) exhibit higher green fluorescenceintensity of fluorescein conjugated- OVA model antigens thanfree model antigen group and large-size cancer vaccines (OVA-ML). The results suggest that small-size cancer vaccines moreFrontiers in Immunology 06significantly enhance the lymph node targeting of tumor antigensin vivo, compared with large-size cancer vaccines and those infree format.Then, cross-presentation of OVA model tumor antigens in vivowere quantitatively investigated by flow cytometry using H-2Kb-SIINFEKL+ in CD11c+ cell populations in lymph node as evaluationindicators (Figure 3B). CD11c is a commonly used as cell markerfor mouse DCs. Herein, H-2Kb-SIINFEKL+ in CD11c+ cellpopulations in lymph node represent cross-presentation of OVAtumor antigens by DC cells. FeMOF-based cancer vaccines in smallsize (OVA-MS) significantly enhance cross-presentation of tumorantigens in vivo, compared with all the other groups, includingsaline group, free OVA group and FeMOF-based cancer vaccines inlarge size (OVA-ML).The lymph node targeting of tumor antigens and theirsubsequent antigen cross-presentation play a very critical role inBAFIGURE 2FeMOF-based cancer vaccines effectively enhance antigen uptake and activation of DCs in vitro. (A) Representative confocal laser scanningmicroscope images of dendritic cells after culture with free F-OVA and vaccines overnight with lysosome staining (scale bar 20mm). (B) Quantitativeanalysis of DCs activation after culture with free OVA and self-assembled vaccines for 1 days. Data in b, n=3 independent samples, one-way ANOVAfollowed by Tukey’s multiple comparisons post hoc test, p<0.05. All data are presented as mean ± SD.frontiersin.orghttps://doi.org/10.3389/fimmu.2023.1328379https://www.frontiersin.org/journals/immunologyhttps://www.frontiersin.orgLi et al. 10.3389/fimmu.2023.1328379inducing tumor antigen-specific immunity. In this study, FeMOF-based cancer vaccines in small size is the most effective among allgroups in enhancing the lymph node targeting of tumor antigensand strengthening their cross-presentation in vivo.3.4 Antitumor effects of FeMOF-basedvaccines in therapeutic mouse E.G7-OVAlymphoma modelTwenty female C57BL/6J mice were randomly divided into fourgroups. E.G7-OVA lymphoma cells (1.2×105 cells/mouse) wereFrontiers in Immunology 07inoculated subcutaneously into the left flank of mice to establishthe therapeutic tumor model (Figure 4A). On days 4, 7, and 10 posttumor inoculation, FeMOF-based cancer vaccines in large size(OVA-ML) and small size (OVA-MS) in 100mL saline weresubcutaneously administrated into the right flank of mice. Inaddition, only saline and free OVA in saline were administratedas control groups. Later, tumor size was continuously measured tostudy the therapeutic effect of tumor vaccines on distant tumors(Figure 4B). Saline group and free OVA group show considerableand rapid tumor growth of E.G7-OVA lymphoma. However, micetreated with FeMOF-based cancer vaccines exhibited the inhibitionin tumor growth, compared with saline and free OVA groups.BAFIGURE 3FeMOF-cancer vaccines in small size significantly enhance lymph node targeting and cross-presentation of tumor antigens in vivo. (A) Representativeconfocal laser scanning microscope images of lymph nodes in mice administrated with free F-OVA and FeMOF-based cancer vaccines with large sizeand small size (OVA-ML and OVA-MS, scale bar 50mm). (B) Representative flow cytometry plots of H-2Kb-SIINFEKL+ in CD11c+ cells population in lymphnodes of mice vaccinated with free OVA and FeMOF-based cancer vaccines (Left). Quantitative analysis of H-2Kb-SIINFEKL+ in CD11c+ cells populationin lymph nodes of mice vaccinated with free OVA and FeMOF-based cancer vaccines (Right). Data in b, right, n=3 independent animals, one-wayANOVA followed by Tukey’s multiple comparisons post hoc test, p<0.05. All data are presented as mean ± SD.frontiersin.orghttps://doi.org/10.3389/fimmu.2023.1328379https://www.frontiersin.org/journals/immunologyhttps://www.frontiersin.orgLi et al. 10.3389/fimmu.2023.1328379Especially, FeMOF-based cancer vaccines in small size (OVA-MS)more effectively inhibited the tumor growth of E.G7-OVAlymphoma than those in large size (OVA-ML). The therapeuticeffects of FeMOF-based cancer vaccines on E.G7-OVA lymphomasuggest that FeMOF is not only a delivery system for OVA modelFrontiers in Immunology 08tumor antigens, but also an effective adjuvant to trigger anti-tumorimmune response.To investigate the underlying antitumor mechanism of FeMOF-based cancer vaccines, the spleens of mice at the endpoint ofantitumor experiments were collected to check CD4+, CD8+ andBCDEAFIGURE 4Antitumor effects of FeMOF-based cancer vaccines in therapeutic mouse E.G7-OVA lymphoma model. (A) Schematic illustration of antitumorexperiments: E.G7-OVA lymphoma cells (1.2×105 cells/mouse) were inoculated subcutaneously into the left flank of female C57BL/6J mice; On days4, 7, and 10 post tumor inoculation, FeMOF-based cancer vaccines were injected into the right flank of mice; Tumor growth was continuouslymeasured. (B) Average tumor growth curves of different vaccines formulations. Data in b, n=5 independent animals, one-way ANOVA followed byTukey’s multiple comparisons post hoc test, p<0.05. All data are presented as mean ± SD. (C-D) Representative flow cytometry plots (C) andpopulations (D) of CD4+, CD8+ and tetramer+CD8+ T cells in spleen at the endpoint. (E) Quantitative analysis of cytokines in spleen at the endpoint.Data in d and e, n=3 independent animals, one-way ANOVA followed by Tukey’s multiple comparisons post hoc test, p<0.05. All data are presentedas mean ± SD.frontiersin.orghttps://doi.org/10.3389/fimmu.2023.1328379https://www.frontiersin.org/journals/immunologyhttps://www.frontiersin.orgLi et al. 10.3389/fimmu.2023.1328379tetramer+CD8+ T cell populations (Figures 4C, D). The CD4+,CD8+ and tetramer+CD8+ T cell populations in splenocytes inFeMOF-based cancer vaccines in large size (OVA-ML) and smallsize (OVA-MS) are significantly higher than saline group and freeOVA group. The average CD4+ and CD8+ T cell populations insmall-size cancer vaccines (OVA-MS) are higher those in in large-size cancer vaccines (OVA-ML), although no significant differenceis observed. Moreover, the cytokines in spleen were determined byELISA assay (Figure 4E). Small-size cancer vaccines (OVA-MS)more efficiently stimulated the secretion of IFN-g and IL-12,compared with other groups.3.5 Antitumor effects of FeMOF-basedvaccines in therapeutic mouse Lewis lungcarcinoma modelFemale C57BL/6J mice were randomly divided into four groups.LLC cells (8×104 cells/mouse) were inoculated subcutaneously intothe left flank of mice to establish the therapeutic tumor model(Figure 5A). On days 4, 7, and 10 post tumor inoculation, FeMOF-based cancer vaccines in large size (dLLC-ML) and small size(dLLC-MS) in 100mL saline were subcutaneously administratedinto the right flank of mice. Saline group and dLLC autologoustumor antigens in free format were used as controls. Later, tumorsizes were monitored to confirm the therapeutic effect of FeMOF-based personalized cancer vaccines towards lewis lung carcinoma inthe distant sites (Figure 5B). As shown in the curves, only dLLCautologous tumor antigens in free format did not inhibit the tumorgrowth of Lewis lung carcinoma, compared with free saline group.While FeMOF-based cancer vaccines encapsulated with dLLCautologous tumor antigens effectively inhibited tumor growth,compared with saline and free dLLC autologous tumor antigensgroups. Moreover, FeMOF-based cancer vaccines in small size(dLLC-MS) more effectively inhibited the tumor growth thanthose in large size (dLLC-ML). In general, FeMOF-based cancervaccines encapsulated with dLLC autologous tumor antigens showFrontiers in Immunology 09the same tendency to inhibit tumor growth as those encapsulatedwith OVA model tumor antigens.To investigate the underlying antitumor mechanism of FeMOF-based cancer vaccines encapsulated with dLLC autologous tumorantigens, the spleens of mice at the endpoint of antitumorexper iments were col lected to analyze CD4+, CD8+,CD44highCD62Lhigh in CD4+, and CD44highCD62Lhigh in CD8+ Tcell populations (Figures 6, 7). The CD4+ and CD8+ T cellpopulations in splenocytes in FeMOF-based cancer vaccines inlarge size (dLLC-ML) and small size (dLLC-MS) are significantlyhigher than saline group and free dLLC autologous tumor antigensgroup (Figure 6). More importantly, the average CD4+ and CD8+ Tcell populations in small-size cancer vaccines (dLLC-MS) are higherthose in in large-size cancer vaccines (dLLC-ML). To furtheranalyze the immunological memory responses induced byFeMOF-based cancer vaccines, the central memory T cells(CD44highCD62Lhigh in CD4+ or CD8+) in the spleens in variousgroups were tested by flow cytometry. The percentage of the centralmemory T cells in mice treated with FeMOF-based cancer vaccines,such as CD44highCD62Lhigh in CD4+ T cells, is much higher thanthat those treated with saline or free dLLC autologous tumorantigens (Figure 7).4 DiscussionDue to their tunable composition, versatile structure, anddiverse functions, metal organic frameworks have attractedincreasing attention in biomedical field, such as drug deliverysystem, cancer therapy, imaging and so on. Metal organicframeworks (MOF) are constructed from the coordinating self-assembly of metal moieties with organic ligands, which can beexogenous or endogenous. In this study, endogenous fumarateligands were employed together with iron ions, one of the mostimportant minerals in humans, to prepare iron-based metal organicframeworks with good biocompatibility. As we mentioned in theintroduction part, fumaric acid is an important intermediateBAFIGURE 5Antitumor effects of FeMOF-based cancer vaccines in therapeutic mouse Lewis lung carcinoma model. (A) Schematic illustration of antitumorexperiments: Lewis lung carcinoma cells (8×104 cells/mouse) were inoculated subcutaneously into the left flank of female C57BL/6J mice; On days4, 7, and 10 post tumor inoculation, FeMOF-based cancer vaccines were injected into the right flank of mice; Tumor growth was continuouslymeasured. (B) Average tumor growth curves of different vaccines formulations. Data in b, n=4 independent animals, one-way ANOVA followed byTukey’s multiple comparisons post hoc test, p<0.05. All data are presented as mean ± SD.frontiersin.orghttps://doi.org/10.3389/fimmu.2023.1328379https://www.frontiersin.org/journals/immunologyhttps://www.frontiersin.orgLi et al. 10.3389/fimmu.2023.1328379product of the citric cycle in the body, which is a source ofintracellular energy in the form of ATP (22). Fumarate is recentlyreported to stimulate interferon secretion and trigger innateimmunity (27), which has the potential to enhance the immuneinfiltration in cold tumors. On the other hand, iron plays a pivotalrole in the innate and adaptive immunity, such as macrophagepolarization, natural killer cells activity, T cells activity and so on(30). Iron deficiency leads to the inhibition of T cells proliferationand antibody immune response (30). Herein, FeMOF built fromiron ions and endogenous fumarate ligands not only serves as adelivery system for tumor antigens, but also acts as an intrinsicadjuvant to enhance the immune response to tumor antigens.Moreover, in this study, the aqueous green synthesis route in icewith no use of toxic organic solvents is adopted to achieve the one-pot synthesis of FeMOF-based cancer vaccines with highencapsulation efficiency larger than 80% and effectively maintainsthe immunogenicity of tumor antigens.FeMOF-based cancer vaccines are promising for bothpredefined personalized antigens vaccines and unidentifiedpersonalized antigens vaccines. In this study, two distinct mousetumor models, including mouse E.G7-OVA lymphoma model withpredefined tumor antigen and Lewis lung carcinoma models withunidentified tumor antigens, were used to evaluate the therapeuticFrontiers in Immunology 10efficacy of FeMOF-based cancer vaccines. Herein, FeMOF efficientlyencapsulates these two personalized tumor antigens, such as OVAmodel tumor antigens and dLLC autologous tumor antigens, withencapsulation efficiency higher than 80%. We demonstrated theconcept of one-pot FeMOF-based cancer vaccines using these twocompletely different types of tumor cells. This approach is expectedto be a universal method that can be applied to other types oftumors simply by replacing tumor cell lysates or designatedtumor antigens.In vitro assay using primary DCs and in vivo analysis ofcytokines in spleen suggest that FeMOF materials exhibit intrinsicadjuvant properties by promoting the secretion of Th1 cytokines,such as IL-12, TNF-a, and IFN-g. IL-12 is a proinflammatorycytokine, which primarily produced by antigen-presenting cells,such as DCs, macrophages, monocytes and so on (31). IL-12exhibits multiple immunomodulatory functions, such as inducingthe differentiation of Th0 into Th1 lymphocytes, increasing thecytolytic activation of NK cells and cytotoxic T lymphocytes, andpromoting the secretion of TNF-a and IFN-g by T cells, all of whichfacilitates to transform the immunosuppressive cold tumors intoimmunologically active hot tumors (31). TNF-a is predominantlyproduced by activated antigen-presenting cells and is a powerfultumoricidal cytokine, as its name describes, which induces theBAFIGURE 6Antitumor mechanism analysis of FeMOF-based cancer vaccines in therapeutic mouse Lewis lung carcinoma model. Representative flow cytometryplots (A) and populations (B) of CD4+ and CD8+ T cells in spleen at the endpoint. Data in b, n=4 independent animals, one-way ANOVA followed byTukey’s multiple comparisons post hoc test, p<0.05. All data are presented as mean ± SD.frontiersin.orghttps://doi.org/10.3389/fimmu.2023.1328379https://www.frontiersin.org/journals/immunologyhttps://www.frontiersin.orgLi et al. 10.3389/fimmu.2023.1328379apoptotic cell death, stimulates the inflammatory response at thetumor sites and inhibits the tumorigenesis (31). IFN-g not onlyexhibits immunomodulatory functions on innate and adaptiveimmune response, but also has direct cytotoxic effects on tumorcells (31). Although these kinds of Th1 cytokines, including IL-12,TNF-a, and IFN-g, are promising in cancer immunotherapy, theirclinical application is associated with short half-life, narrowtherapeutic window, severe dose-limiting toxicities, anddifficulties in large-scale manufacturing (31). In this study,FeMOF adjuvant materials may induce antigen-presenting cells tosecrete Th1 cytokines in situ, stimulate the secretion of Th1cytokines in immune organs and trigger systemic activation ofantitumor immunity, which provides another way to use cytokinesin cancer immunotherapy.FeMOF-based cancer vaccines based on different tumorantigens effectively trigger T-cells immune response in differentkinds of tumor-bearing mice, including E.G7-OVA lymphomamodel and Lewis lung carcinoma model. Vaccination usingFeMOF-based cancer vaccines efficiently increase the cellpopulations of typical T lymphocytes in immune organs, such asCD4+, CD8+ cells and so on. CD4+ T cells play a prominent role inadaptive immunity, which are traditionally considered to providehelp for CD8+ cells to trigger antitumor immune response, andrecently reported to also own direct antitumor capacity (32). CD8+T cells, often called cytotoxic T lymphocytes, are the major driversof antitumor immunity and have the capacity to selectively detectFrontiers in Immunology 11and eliminate cancer cells. In E.G7-OVA lymphoma model, OVAtumor antigens-specific tetramer+CD8+ T cells have also beenanalyzed, and the results suggest that FeMOF-based cancervaccines resulted in the increase in tumor antigens-specific CD8+T cells populations. On the other hand, in Lewis lung carcinomamodel, the central memory T cells (CD44highCD62Lhigh in CD4+ orCD8+) have been quantified, which suggests that FeMOF-basedcancer vaccines may enhance immune memory.Due to time and space limitations, the present study focuses onevaluating the anti-tumor efficacy of FeMOF-based cancer vaccineswith tailored morphology using two different tumor models, andtheir effects on immune response in spleen and lymph. The effects ofcancer vaccines on the tumor microenvironment are not involved.According to existing literature reports, we have reason to believethat the rationally designed cancer vaccines may have a great impacton the tumor microenvironment, such as the infiltration of tumor-specific T cells, the macrophages polarization towards M1 type, theinhibition of regulatory T cells (Treg) and myeloid-derivedsuppressor cells (MDSC) and so on (33–35). Further research onthe above contents will be conducted in the future.5 ConclusionsIn summary, a rapid one-pot synthetic route has been developedto synthesize personalized cancer vaccines using model antigen orBAFIGURE 7Antitumor mechanism analysis of FeMOF-based cancer vaccines in therapeutic mouse Lewis lung carcinoma model. Representative flow cytometryplots (A) and populations (B) of CD44highCD62Lhigh in CD4+ and CD8+ T cells in spleen at the endpoint. Data in b, n=4 independent animals, one-way ANOVA followed by Tukey’s multiple comparisons post hoc test, p<0.05. All data are presented as mean ± SD.frontiersin.orghttps://doi.org/10.3389/fimmu.2023.1328379https://www.frontiersin.org/journals/immunologyhttps://www.frontiersin.orgLi et al. 10.3389/fimmu.2023.1328379autologous tumor antigens based on the coordination interactionbetween Fe3+ ions and endogenous fumarate ligands. Herein, Fe-based metal organic framework can effectively encapsulate tumorantigens with high loading efficiency >80%, and act as both deliverysystem and adjuvants for tumor antigens. By adjusting the synthesisparameters, the morphology of the obtained cancer vaccines is easilytailored from microscale to nanoscale. When cocultured withantigen-presenting cells, nanoscale cancer vaccines more effectivelyenhance antigen uptake and Th1 cytokine secretion than microscaleones. Nanoscale cancer vaccines more effectively enhance lymphnode targeting and cross-presentation of tumor antigens, mountantitumor immunity, and inhibit the growth of established tumorin tumor-bearing mice, compared with microscale cancer vaccines.Our approach to developing personalized cancer vaccines has thepotential to be applied to other tumor types by replacing tumor celllysates or designated tumor antigens.Data availability statementThe raw data supporting the conclusions of this article will bemade available by the authors, without undue reservation.Ethics statementThe animal study was approved by The Animal EthicsCommittee of National Institute for Materials Science (NIMS),Japan. The study was conducted in accordance with the locallegislation and institutional requirements.Author contributionsXL: Conceptualization, Data curation, Formal analysis, Fundingacquisition, Investigation, Methodology, Project administration,Resources, Writing – original draft, Writing – review & editing.SH: Methodology, Writing – review & editing. ME: Methodology,Frontiers in Immunology 12Writing – review & editing. NS: Methodology, Writing – review &editing. NH: Methodology, Supervision, Writing – review & editing.FundingThe author(s) declare financial support was received for theresearch, authorship, and/or publication of this article. Wegratefully acknowledge the financial support from Japan Society forthe Promotion of Science (JSPS, KAKENHI Grant No. 23K04555 and22K20517), Takeda Science Foundation, and NIMS funds. This workwas supported in part by “Advanced Research Infrastructure forMaterials and Nanotechnology in Japan (ARIM)” of the Ministry ofEducation, Culture, Sports, Science and Technology (MEXT), Japan.Proposal Number JPMXP1223NM5201.AcknowledgmentsWe thank Dr. T. Takemura, Dr. Y. Shirai, Dr. A. Yamamoto,Dr. X.L. Li, and Ms. S. Kohara for their kind assistance duringthe experiments.Conflict of interestThe authors declare that the research was conducted in theabsence of any commercial or financial relationships that could beconstrued as a potential conflict of interest.Publisher’s noteAll claims expressed in this article are solely those of the authorsand do not necessarily represent those of their affiliated organizations,or those of the publisher, the editors and the reviewers. Any productthat may be evaluated in this article, or claim that may be made by itsmanufacturer, is not guaranteed or endorsed by the publisher.References1. Sahin U, Tureci O. 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